Alpha-1 antitrypsin is a protein which is composed of 394 amino acid residues, has a molecular weight of approximately 50,000 daltons (Da), and is present in the blood of mammals. Alpha-1 antitrypsin is one of main blood proteins whose blood concentration amounts to approximately 2 mg/mL (Robin W. C. et. al., Nature, 298, 329-334, 1982), and is also referred to as an alpha-1 protease inhibitor. Alpha-1 antitrypsin has at least about 100 naturally occurring alleles, and its phenotypes are classified into categories A to Z according to the isoelectric focusing (IEF) profiles. Among these, the most abundant M-type allele is known to be present in the blood of most humans and to have at least approximately 75 different isoforms (Brantley, M. et. al., Am. J. Med., 84(suppl. 6A), 13-31, 1988), and maintains a main function as a protease inhibitor.
In general, approximately 90% of alpha-1 antitrypsin isoforms are known to be present as five PiM subtypes such as M1, M2, M3, M4, and M5. Among these, M1, M2 and M3 are distributed at approximately 67%, approximately 16% and approximately 11%, respectively (Jan-Olof Jeppsson and Carl-Bertil Laurell, FEBS Lett., 231, 327-330, 1988). Among these, the M2 and M3 subtypes are known to have histidine (His) and arginine (Arg) at a 101st position from the N-terminus, respectively, and these amino acid difference is known to have no effect in innate activities of alpha-1 antitrypsin.
Alpha-1 antitrypsin is a glycoprotein which is glycosylated at 3 sites (Mega, T. et. al., J. Biol. Chem., 255, 4057-4061, 1980). X-ray crystal structure shows that it is composed of 3 beta sheets and 8 alpha helices like other protease inhibitors (serpins) present in blood (Elliot P. R., et. al., JMB, 275, 419-425, 1998). Alpha-1 antitrypsin functions to inhibit various kinds of proteases in the body, and its main in vivo function associated with diseases currently known in the related art is to inhibit neutrophil elastase activities (Beatty et. al., J. Biol. Chem., 255, 3931˜3934, 1980). The deficiency of alpha-1 antitrypsin causes severe diseases such as pulmonary emphysema in which pulmonary functions are impaired due to the decomposition of elastin. Also, there is a clinical report showing that modified proteins of alpha-1 antitrypsin are not normally secreted by the liver, but accumulate in the liver, which leads to the onset of hepatocirrhosis.
In recent years, several products extracted from the human blood have been approved by US Food and Drug Administration (FDA), and have been on the market as therapeutic agents to treat alpha-1 antitrypsin deficiency. Representative examples of the products include Prolastin® (commercially available from Talecris Plasma Resources Inc), Aralast™ (commercially available from Baxter Inc), and Zemaira® (commercially available from CSL Behring Inc), which are generally administered at a dose of 60 mg/kg to a human body by intravenous injection at intervals of one week. Therefore, the protein should be administered weekly to an adult patient at a large dose of 4 to 5 g over a long period of time.
According to Data Monitor (DMHC2364) analysis, there are probably 200,000 patients with genetic problems associated with alpha-1 antitrypsin in the US and Europe, but only some of these patients have been treated because proper diagnoses are not made. All the products currently developed as medical purpose are alpha-1 antitrypsin extracted from human blood. Such alpha-1 antitrypsin extracted from human blood may have a risk of including viruses derived from the human body which may cause diseases fatal to humans such as human immunodeficient virus (HIV), hepatitis B virus, or hepatitis C virus, even if it is completely eliminated during a production procedure. Even when alpha-1 antitrypsin is subjected to a blood screening test for the detection of several pathogens and a virus inactivation procedure, it is impossible to eradicate rare pathogens that are not yet known. Therefore, there is always a risk of infection by use blood extracted alpha-1 antitrypsin from unknown pathogens in the human body. Also, the stable supply of uncontaminated blood used to produce a commercially required amount of alpha-1 antitrypsin has been problematic.
As an alternative to solve the aforementioned problems, recombinant DNA technology can be used to develop alpha-1 antitrypsin as a therapeutic agent. Therefore, the recombinant DNA technology has been continuously researched, but there has been no commercially available recombinant alpha-1 antitrypsin yet due to various limiting factors.
Human alpha-1 antitrypsin is known to have 3 N-glycan moieties as it is glycosylated at 3 sites (asparagine at a 46th position, asparagine at an 83rd position, and asparagine at a 247th position). Since alpha-1 antitrypsin produced by a recombinant DNA method using a microorganism such as E. coli is not glycosylated, it is known to have a short in vivo half-life when administered to the body (Karnaukhova et. al., Amino Acids, 30, 317-332, 2006, Garver Jr. et. al., Proc. Natl. Acad. Sci. USA., 84, 1050-1054, 1987). To solve this problem and also effectively produce a large amount of alpha-1 antitrypsin, research has been conducted by the expression of alpha-1 antitrypsin in plants. However, it was reported that although the recombinant alpha-1 antitrypsin expressed in plants contained plant-derived glycosylation, it had a shorter half-life in the body than the human alpha-1 antitrypsin (Huang et. al., Biotechnol. Prog., 17, 126-133, 2001).
To increase the half-life of alpha-1 antitrypsin in the body, Cantin et. al. reported a fusion protein by conjugating a polyethylene glycol to a cysteine residue of alpha-1 antitrypsin expressed in microorganisms (Cantin et. al., Am. J. Respir. Cell. Mol. Biol., 27, 659-665, 2002). The article demonstrated that when a polyethylene glycol having a molecular weight of 20 to 40 kDa was conjugated to the cysteine residue of alpha-1 antitrypsin expressed in a microorganism, the conjugated alpha-1 antitrypsin had an increased half-life in the body, compared with the alpha-1 antitrypsin expressed in the microorganism, resulting in the substantially similar half-life to that of the human alpha-1 antitrypsin. However, when a polyethylene glycol is conjugated to a protein, various heterogeneous reaction products can be formed by chemical side reactions. As a result, additional processes are required to remove the heterogeneous reaction products. Also, because there is no N-glycan moieties in a PEG conjugated alpha-1 antitrypsin, this may cause immunogenicity problem by the exposed amino acid sequences when treated for human beings.
Alpha-1 antitrypsin derived from animal cells is known to have substantially the same half-life in the body as human alpha-1 antitrypsin (Garver Jr. et. al., Proc. Natl. Acad. Sci. USA., 84, 1050-1054, 1987). Therefore, a method of producing alpha-1 antitrypsin having a structure similar to the human alpha-1 antitrypsin in animal cells may be preferable. In spite of the advantage of animal cell derived alpha-1 antitrypsin, the production of alpha-1 antitrypsin using the animal cells has a problem in that it is generally more expensive than a method of producing alpha-1 antitrypsin in microorganisms.
Meanwhile, technology of adding a glycosylation site to a loop region of alpha-1 antitrypsin has been suggested to increase the in vivo half-life of alpha-1 antitrypsin. In general, it can be hypothesized that when a protein expressed in animal cells is glycosylated, the protein can be considered to have an increased half-life in the body due to the increased hydrodynamic volume of the glycosylated protein when administered to the human body, compared with the proteins which are not glycosylated. However, as can be seen from examples of erythropoietins, the alteration or addition of a glycosylation site to a physiologically active protein has a great influence on the in vivo half-life of the protein depending on the glycosylation positions (Eliott et. al., Nat. Biotechnol., 21, 414-421, 2003). Therefore, when a glycosylation site is added to alpha-1 antitrypsin in order to increase the in vivo half-life and physiological stability, one has to prove extensively to which position(s) of alpha-1 antitrypsin a glycosylation site is added.
In conclusion, there have been various methods attempted to prepare alpha-1 antitrypsin using recombinant DNA technology in order to enhance in vivo stability of alpha-1 antitrypsin. However, such existing methods are not suitable for the development of alpha-1 antitrypsin as a medicine due to the various problems as described above. Therefore, there is an urgent need for a new method to develop a recombinant alpha-1 antitrypsin having excellent stability in the body.